|Year : 2021 | Volume
| Issue : 1 | Page : 141-145
Defining the normal range for screening panel of neutrophil function tests in healthy adult Indian participants
Shivanand S Pattan, Kishore G Bhat, Vijay M Kumbar, Manohar S Kugaji, Geeta Pattar
Central Research Laboratory, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgavi, Karnataka, India
|Date of Submission||17-Jul-2020|
|Date of Acceptance||26-Nov-2020|
|Date of Web Publication||09-Feb-2021|
Dr. Kishore G Bhat
Central Research Laboratory, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Bauxite Road, Belgavi - 590 005, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Neutrophils, the most abundant white blood cells are known to play a major role in the fundamental physiological response to tackle with microbial infections via different mechanisms. A series of assays have been developed that mimic neutrophil functions both in vivo and in vitro to look at specific deficiencies of neutrophil function. There is need to develop method to have defined normal range for these tests to differentiate between health and disease. We aim to define normal range for panel of screening assays in healthy adult Indian participants.
Materials and Methods: The study included 30 male and 30 female adult healthy participants. A volume of 10 ml of blood was collected from each participant. Four different assays that included chemotaxis, phagocytosis, intracellular killing (candidacidal assay), and nitroblue tetrazolium (NBT) test were performed. For defined range was established for all the assays. Simultaneously controls were set up for all the tests.
Results: For NBT assay, the mean values for control and test were 13.58 and 58.90 respectively. The mean values for the control and test for phagocytosis assay were 1.77 and 4.49. Similarly, the values for candidacidal assay were 11.67% and 34.98. Finally, the values for chemotaxis test were 2.13 cm for Formyl-Methionyl-Leucyl-Phenylalanine, 0.54 cm for participant's serum and 0.48 cm for the medium.
Conclusion: Based on the data generated, the values produced in the present investigation should be considered as normal range for adult population between the age group of 25–50 years for the protocols followed in the study.
Keywords: Chemotaxis, intracellular killing, neutrophil function, nitroblue tetrazolium, phagocytosis
|How to cite this article:|
Pattan SS, Bhat KG, Kumbar VM, Kugaji MS, Pattar G. Defining the normal range for screening panel of neutrophil function tests in healthy adult Indian participants. Indian J Health Sci Biomed Res 2021;14:141-5
|How to cite this URL:|
Pattan SS, Bhat KG, Kumbar VM, Kugaji MS, Pattar G. Defining the normal range for screening panel of neutrophil function tests in healthy adult Indian participants. Indian J Health Sci Biomed Res [serial online] 2021 [cited 2021 Feb 27];14:141-5. Available from: https://www.ijournalhs.org/text.asp?2021/14/1/141/308954
| Introduction|| |
Neutrophils are the most abundant white blood cells in circulation. They co-ordinate and execute one of the most fundamental physiologic responses, i.e., inflammation. They are endowed with antimicrobial mechanisms that make them the preeminent microbe killers of the immune system. Neutrophils have a high potency and efficiency to sense and eradicate microbial infections and individuals with a neutrophil deficiency are more susceptible to microbial and fungal infections. They engage and kill microbes at sites of infection via a number of different mechanisms including chemotaxis, phagocytosis, release of reactive oxygen species, and granular proteins and production and liberation of cytokines., Recent evidence shows the neutrophils to be highly heterogeneous population with different functional phenotypes in inflammation and cancer.,, Malfunctioning of neutrophils results in chronic inflammation which is mainly to the accumulation of cytotoxic substances and are associated with severe pathologies such as cystic fibrosis, chronic obstructive pulmonary disease, and rheumatoid arthritis.,
The multifaceted role of neutrophils in immune regulation indicates that no picture of immune response can be complete without proper understanding of the neutrophil and its functions. Hence, to localize specific deficiencies of neutrophil function, a series of assays have been developed that mimic these functions both in vivo and in vitro. Usually, a preliminary screening of several functions is performed to pinpoint the abnormalities before subjecting the sample to a specific functional assay.
It is of utmost importance for any test to have a normal range so that healthy status can be differentiated from disease. Literature survey has shown that for all the assays of neutrophil function, there is no defined normal range in Indian subjects and one is compelled to depend on the western parameters as standard. With this background, we decided to take up an investigation to define the normal range for a panel of screening assays in healthy adult Indian participants.
| Materials and Methods|| |
The study included 30 male and 30 female adult healthy participants between the age group of 25–50 years. This investigation was part of a funded project (2017–2018) from Rajiv Gandhi University of Health Sciences (RGUHS) titled “Phagocytic function tests-Establishing guidelines and designing normal levels in blood and saliva of healthy adult subjects.” The study was approved by institutional ethical committee and a formal written consent was obtained from each participant before enrollment. Only those subjects who were apparently healthy, were not anemic, had a normal total and differential white blood cell count, were not tobacco users, nonalcoholic and were not suffering from diabetes mellitus or any other systemic illness were included in the study.
Ethical Clearance was obtained from Maratha Mandal's NGH Institute of Dental Sciences & Research Centre, Belgaum, Institutional Ethical Committee with Ref No. 629 dated 10.07.2017.
A volume of 10 ml of blood was collected from each subject in two vacutainers containing ethylenediamine tetraacetic acid as anticoagulant. All samples were processed within 4 h of collection. Four different assays that included chemotaxis, phagocytosis, intracellular killing (candidacidal assay), and nitroblue tetrazolium (NBT) test were performed on each sample. While NBT test was carried out with whole blood, other three assays were performed with purified white blood cells (WBCs).
Each blood sample was diluted with equal quantity of Hank's balanced salt solution and was used for further processing on WBC separation with gelatin solution., For this purpose, 3 ml of diluted blood was mixed with 2 volumes of gelatin 0.5% (w/v, prepared in 0.9% NaCl), mixed properly and incubated at room temperature for 45 min. The cell rich supernatant was then transferred to another tube and centrifuged at 1500 rpm for 5 min to get the cell pellet. This was washed three times with sterile phosphate buffered saline (PBS) (pH 7.2) to remove remnants of gelatin. The presence of WBCs in the preparation was confirmed by microscopy.
A qualitative NBT test was performed on the whole blood of each subject. The test was set up in two tubes, one for stimulated cells and the other one for unstimulated cells. In one tube, 100 μl of blood was mixed with 100 μl RPMI, 100 μl of NBT (0.35 w/v prepared in 0.34% sucrose, diluted in PBS just before use) and 100 μl of endotoxin (prepared in house from Escherichia More Details coli). In the second tube, endotoxin was replaced with additional 100 μl of RPMI. Both the tubes were incubated for 20 min at 37°C and further 20 min at room temperature. The tubes were mixed properly and a thin smear was prepared from each tube, stained with giemsa and observed microscopically under oil immersion (×100). At least 200 neutrophils were counted and cells containing bluish black formazan granules were considered as stimulated by endotoxin [Figure 1]. The ratio of stimulated versus unstimulaed cells were expressed as percentage.
|Figure 1: Nitroblue tetrazolium assay: Single neutrophil showing intracellular formazan granules|
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The remaining assays were performed using the purified WBC preparations. Phagocytosis function was tested using heat killed Candida albicans. To one tube, 100ul each of Hanks solution, pooled normal human serum, heat killed Candida suspension and WBC suspension were added. In a second tube, which acted as a negative control, serum was replaced with additional 100 μl of Hanks solution. The tubes were mixed well and incubated at 37°C for 30 min. The tubes were centrifuged at 1500 rpm for 5 min and supernatant was discarded. A smear was prepared from the sediment, air dried and stained with Giemsa stain. The smears were observed under microscope, 200 neutrophils were examined and number of Candida ingested per cell was counted [Figure 2]. After calculation, the number of Candida was expressed as mean particle number (MPN).
Candidacidal assay was carried out to test for intracellular killing function. To one tube, 100 μl of WBC suspension, 100 μl of Hanks solution, 100 μl of autologous plasma, and 100 μl of live Candida cells were added. To another tube which acted as control, WBC suspension was replaced with 100 μl of Hanks solution. The tubes were incubated in a 37°C water bath for 1 h with intermittent shaking. At the end of the incubation, 100 μl of 2.5% sodium deoxycholate was added to each tube and mixed well. This was followed by addition of 2 ml of 0.01% methylene blue, mixed and centrifuged at 2000 rpm for 10 min. Supernatant was discarded leaving about 0.25 ml of the suspension. This was mixed well, a small drop was transferred to a Neubauer counting chamber and at least 300 Candida cells were counted. The proportion of dead cells was determined by counting the cells stained with methylene blue versus unstained cells [Figure 3].
|Figure 3: Intracellular killing: Colorless viable Candida and blue colored dead Candida|
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Chemotactic activity of each sample was tested against a known chemoattractant Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) under agarose. On a clean glass slide (25 mm × 75 mm), 5 ml of 1.2% agarose solution containing MEM medium, heat inactivated pooled human serum, and sodium bicarbonate was poured and allowed to solidify. Series of wells of 3 mm diameter were cut into the agarose using a template. The central well was loaded with fMLP and the peripheral wells contained WBC cells, buffer and a separate control was set up where chemoattractant was replaced with subject's serum. The slides were kept in a humidified chamber and incubated at 37°C for 2 h. Slides were then immersed in methanol for 30 min followed by fixation with formalin for 30 min. Agarose layer was gently removed and the cells on the slide were stained with Giemsa. Then the linear distance traveled by the cells under the effect of FMLP was measured under the microscope using an oculometer [Figure 4]. Spontaneous migration toward the well containing buffer was also noted to calculate chemotactic index.
| Results|| |
To analyze the data, we calculated the mean of control and test values of each of the screening assays. For NBT assay, the mean values for control (unstimulated) and test (stimulated) were 13.58 (range 8–38) and 58.90 (range 42–76), respectively. The mean values for the control and test for phagocytosis assay were 1.77 (range 1–3) and 4.49 (range 3–7). Similarly, the values for candidacidal assay were 11.67% with a range of 09%–16% and 34.98% with a range of 32%–42%. Finally, the values for chemotaxis test were 2.13 cm (1.2–2.9 cm) for FMLP, 0.54 cm (0.4–0.8 cm) for participant's serum and 0.48 cm (0.4–0.5 cm) for the medium which was used for the assay. In addition, we calculated median, 25% percentile and 75% percentile values for control and test readings of each assay. The details are shown in [tTable 1].
|Table 1: The mean, mode and median values of different neutrophil function assays in both the genders together|
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We also made a comparison between the mean values of each assay in males and females. However, the readings were almost similar for both the categories without any significant difference [Table 2].
|Table 2: The mean values of different neutrophil function assays in both the genders separately|
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| Discussion|| |
This is the first study of its kind from India wherein the screening assays for neutrophil function are performed in adult healthy controls to define the normal range. In our center, we have been performing the neutrophil function tests for the last several years in different diseases and also to assess the immunomodulatory activities of different synthesized and natural products.,, We are dependent on the values from the western subjects as basis of normal range to assess the technicality and to evaluate the performance of the results of our assays. Literature survey revealed that no study has been conducted in our country to assess the normal levels in our own subjects of different ages.
The literature also shows that there is no consensus about the protocol to be followed for these screening assays and different laboratories have been following different modifications.,,, Irrespective of the method adopted, every test must have a normal control set up with each assay. However, to know whether the controls are within the accepted range or not, it is necessary to define normal range for each assay for different age group in the population of a specific geographic location. In this study, we have adopted most commonly performed protocols to define normal range for the neutrophil function tests.
Several factors need to be considered when performing the assays for neutrophil function. Apart from the methodology to be followed, the type of anticoagulant used for collection of blood, method used for the purification of WBCs, the concentration of each reagent used and the duration of exposure of neutrophils to assay conditions all play a part in deriving the values for the normal range.
We compared our readings with the values from several established protocols on which our methodology is based.,, In NBT assay, <10% normal stimulated cells should contain blue granules. There is a wide variation in the percentage of stimulated cells based on the reagent used for activating neutrophils. For E. coli endotoxin stimulated cells (as used in our assays), the normal readings should be around 24%–63% which is slightly lower than values of our assays. Three subjects showed higher values in control tests (28%, 28% and 38%); these variations could be due to the presence of subclinical infection in these subjects which resulted in stimulation of neutrophils.
In phagocytosis assay, normal neutrophils should contain from no Candida to five or more per cell with a MPN of 4. Our readings are also similar to the values. In candidacidal assay, normal range of killing may vary from laboratory to laboratory but should be between 20% and 35% and the control values which reflect the viability of Candida culture should be around 10%–12%. Our values are almost similar to these readings.
Chemotaxis assays can be performed either by using modified Boyden's chamber or agarose overlay method. Due to the difficulties in setting up the chamber and reproducibility issues, we have been using agarose overlay method in our laboratory. Even with the same basic agarose method, different laboratories have shown different normal range; this variation is mainly due to the difference in the diameter of wells and time permitted for the cells to migrate. Our protocol which is based on the procedure of Metcalf et al. has similar readings as that of the original investigator.
Based on the data generated in the present investigation, we feel that the values produced should be considered as normal range for adult population between the age group of 25–50 years for the protocols followed in the study. Similar such attempts should be made to evaluate the normal ranges in different age groups from India to prepare the guidelines.
Based on the data generated, the values produced in the present investigation should be considered as normal range for adult population between the age group of 25–50 years for the protocols followed in the study.
The authors would like to thank advanced research committee, RGUHS, Bengaluru, Karnataka, for having provided the financial support for this project.
The authors also would like to thank Dr. Ramakant Nayak, Principal, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgavi for providing necessary infrastructure and facility to carry out this project work.
Financial support and sponsorship
The project has been funded by Advanced Research grant, Rajiv Gandhi University of Health Sciences.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]